Anti-tuberculosis (TB) drugs have diverse abilities to penetrate the different host tissues and cell types in which infecting Mycobacterium tuberculosis (Mtb) bacilli are located during active disease. This is important since there is increasing evidence that the respective lesion- penetrating properties of the frontline TB drugs appear to correlate well with their specific activities in standard combination therapy. Together, these observations suggest that rational efforts to discover novel treatment-shortening drugs and drug combinations should incorporate knowledge about the comparative abilities of both existing and experimental anti-TB agents to access bacilli in defined physiological states at different sites of infection, as well as avoid elimination by efflux or inactivation by host and bacterial metabolism. In turn, this raises a key question: how does the occupation of the host macrophage impact exposure of Mtb to known will engineer cell and mycobacterial reporters for use in combination with advanced analytical technologies to investigate intrabacillary drug metabolism and pharmacokinetics (DMPK) in axenic Mtb culture and during macrophage infection. This fully collaborative proposal will shift the focus of traditional pharmacokinetic-pharmacodynamic analyses away from bioavailability toward targeting drug exposure as critical criterion for drug development and the design of dosing strategies. Moreover, in developing techniques to determine DMPK data for the unit of infection comprising host macrophage and infecting bacillus, we will provide critical evidence of the benefits of including these analyses at an early stage in the drug screening algorithm. and experimental drugs? To address this problem, we will engineer cell and mycobacterial reporters for use in combination with advanced analytical technologies to investigate intrabacillary drug metabolism and pharmacokinetics (DMPK) in axenic Mtb culture and during macrophage infection. This fully collaborative proposal will shift the focus of traditional pharmacokinetic-pharmacodynamic analyses away from bioavailability toward targeting drug exposure as critical criterion for drug development and the design of dosing strategies. Moreover, in developing techniques to determine DMPK data for the unit of infection comprising host macrophage and infecting bacillus, we will provide critical evidence of the benefits of including these analyses at an early stage in the drug screening algorithm.